The objective of this project is the research and development of suitable bioanalytical methods to: (1) establish the structure and purity of potential anti-AIDS agents and new antiviral drugs, (2) determine the physical, chemical and biochemical properties, including octanol-water partition coefficients, of these compounds and their metabolites, and (3) measure these drugs and their metabolites in biological samples to elucidate pharmacology and to determine pharmacokinetics. High-performance liquid chromatography (HPLC) and mass spectrometry are the emphasized techniques. The Phase II drug 2'-b-fluoro-2',3'-dideoxyadenosine (F-ddA, lodenosine) and its deaminated anti-HIV-active metabolite 2'-b-fluoro-2',3'-dideoxyinosine (F-ddI) remain the compounds of primary interest. Validated analytical strategies employing reversed-phase HPLC have been used for both the routine and ultrasensitive measurement of F-ddA in HIV-infected human biological fluids. The human metabolism, distribution and pharmacokinetics of oral F-ddA, in both capsule and liquid formulation, have been determined in conjunction with a two-part Phase I clinical trial of this agent in adult AIDS patients. Oral F-ddA given as a component of combination antiretroviral therapy is fully bioequivalent with F-ddA given as monotherapy. Direct fluorogenic derivatization of cellular extracts in conjunction with paired-ion HPLC has been employed for the nonradiochemical measurement of low picomole amounts of intracellular F-ddATP, the active metabolite of both F-ddA and F-ddI. F-ddATP can be measured in periperal blood mononuclear cells from patients treated with F-ddA, but sufficient data is not available to correlate with observed anti-HIV activity. The toxicity, metabolism and biochemical pharmacology of 2'-fluoro-2-deoxyadenosine are under investigation to determine the role of this trace constituent in the toxicity profile of lodenosine. Lipophilic prodrugs of F-ddI activated by adenosine deaminase (ADA) also continue under investigation as potential agents for the treatment of HIV sequestered in the central nervous system (CNS). A physiological pharmacokinetic model has been constructed to study the disposition of selected ADA-activated F-ddI prodrugs by using F-ddA as a model compound. This model is being used to investigate the effects of various physiological and biochemical processes with emphasis on prodrug gastrointestinal absorption, blood-brain-barrier penetration into the CNS, and metabolic activation. Concentration versus time data in plasma and brain following intravenous and oral administration of F-ddA in rats, monkeys and humans are being used for model validation and interspecies scaling. AIDS Title: The Analytical Chemistry of Anti-AIDS Agents
